The ISME Journal (2010) 4, 144–146 & 2010 International Society for Microbial Ecology All rights reserved 1751-7362/10 $32.00 www.nature.com/ismej SHORT COMMUNICATION Identification of genes for dimethyl sulfide production in in the gut of Atlantic Herring (Clupea harengus)

Andrew RJ Curson, Matthew J Sullivan, Jonathan D Todd and Andrew WB Johnston School of Biological Sciences, University of East Anglia, Norwich, UK

Phytoplankton are the primary producers of the sulfur-containing compatible solute dimethylsulfo- niopropionate (DMSP). These cells are consumed by mesozooplankton, which, in turn, may be eaten by marine vertebrates. From the gut of one such animal, the Atlantic Herring Clupea harengus,we isolated strains of the c- Pseudomonas and Psychrobacter that grew on DMSP as sole carbon source, and which produced the environmentally important sulfurous volatile dimethyl sulfide (DMS). In both bacterial genera, this ability was because of the previously identified gene dddD, which specifies an enzyme that liberates DMS from DMSP. DMS production was stimulated by pre-growth of cells on the substrate DMSP. This is the first identification of DMSP-degrading bacteria and their relevant genes in the gut microflora of any vertebrate. The ISME Journal (2010) 4, 144–146; doi:10.1038/ismej.2009.93; published online 27 August 2009 Subject Category: geomicrobiology and microbial contributions to geochemical cycles Keywords: dddD; dimethyl sulfide; dimethylsulfoniopropionate; Herring; Pseudomonas; Psychrobacter

Microbial dimethyl sulfide (DMS) production is bacterial taxa, having been acquired by horizontal very important in the global sulfur cycle, being the gene transfer (Todd et al., 2007, 2009b). major route for the transfer of sulfur from oceans to Dimethylsulfoniopropionate-catabolizing bacteria atmosphere and then back to the land (Sievert et al., have been isolated from several DMSP-rich environ- 2007). Further, by acting as cloud condensation ments, including corals, algal blooms, copepods nuclei, DMS oxidation products induce cloud (Acartia tonsa) and intertidal salt marshes (Tang formation, reflecting more sunlight to space and et al., 2001; Yoch, 2002; Raina et al., 2009). Here, we affecting the climate (Vallina and Simo´, 2007). describe, for the first time, the isolation of such Dimethyl sulfide is produced from dimethylsul- bacteria from fish and identify the relevant ddd foniopropionate (DMSP), a hugely abundant (109 genes. tons per annum) compatible solute made by many The Atlantic Herring (Clupea harengus) feeds marine phytoplankton and macroalgae, and a few on copepod crustaceans, which in turn consume angiosperms (Sievert et al., 2007). Microbes convert DMSP-containing phytoplankton. We sampled the DMSP to DMS by at least three completely different intestine of one such fish, obtained from the North enzymes that cleave DMSP. One of these, DddL, is Sea, using a series of enrichments in DMSP-contain- a DMSP lyase, forming acrylate plus DMS from ing liquid medium, before plating on minimal agar DMSP, and has thus far only been found in marine plates with 5-mM DMSP as the sole carbon source. a-proteobacteria (Curson et al., 2008). Another After 3 days at 28 1C, two different colony types enzyme, DddP, with no sequence similarity to DddL, were identified. Using the universal bacterial pri- occurs in a-proteobacteria and also in some fungi, mers 27F and 1492R, their 16S rRNA genes were but its mechanism of action is unknown (Todd et al., cloned and then sequenced; the closest (99% 2009a). The third system involves an enzyme of identical) relatives were Pseudomonas guineae Class III acyl CoA transferases, encoded by dddD, LMG 24017 (Bozal et al., 2007) and Psychrobacter which releases DMS from DMSP. This gene occurs arenosus R7T (Romanenko et al., 2004). Isolates of not only in marine g-proteobacteria, but also in other both these genera have been obtained from guts of other fish (Hovda et al., 2007) and some DMS- producing strains have been reported (Ansede et al., Correspondence: AWB Johnston, School of Biological Sciences, 2001). University of East Anglia, Earlham Road, Norwich, Norfolk NR4 The newly isolated Pseudomonas and Psychro- 7TJ, UK. bacter strains were termed J465 and J466, respec- E-mail: [email protected] Received 25 May 2009; revised 13 July 2009; accepted 20 July tively. They were assayed for DMSP-dependent 2009; published online 27 August 2009 DMS production (Ddd phenotype), by gas chroma- Isolation of DMS-producing bacteria from Herring ARJ Curson et al 145 tography as in Curson et al. (2008). Both produced and Pseudomonas J465 were constructed in the large amounts of DMS, whose levels were signifi- wide host-range cosmid pLAFR3. These cosmids cantly enhanced when cells were pre-grown with were mobilized into Escherichia coli and screened 5-mM DMSP. Thus, as in other bacteria (Yoch, 2002; for any that conferred DMS production to this Todd et al., 2007), their Ddd þ phenotype is host (Curson et al., 2008). One such cosmid was inducible (Figure 1). Strains J465 and J466 were identified from each library and their inserts examined for growth on acrylate and 3-hydroxy- (of approximately 34 kb (J465) or 21 kb (J466)) were propionate, as some other DMSP-catabolizing spe- sequenced. Both contained homologues of dddD, cies can use these compounds (Yoch, 2002), and this which is responsible for the initial step in DMSP latter molecule is one of the earliest catabolites catabolism. The closest homologues to the DddD of formed after the action of DddD on DMSP (Todd Psychrobacter J466 and Pseudomonas J465 were in et al., 2009b). Pseudomonas J465 did not grow on the a-proteobacterium Rhodobacterales HTCC2083 either compound, but Psychrobacter J466 grew on (68% identical) and the g-proteobacterium Marino- 3-hydroxy-propionate as the sole carbon source. bacter sp. ELB17 (76%), respectvely. In addition to Acrylate and 3-hydroxy-propionate can also induce dddD, several neighboring genes in Psychrobacter DMS production in some bacteria (Yoch, 2002; Todd J466 and Pseudomonas J465 resembled those that et al., 2009b); this was also the case for Psychro- occur near dddD in Marinomonas MWYL1 (Todd bacter J466, but not Pseudomonas J465 (Figure 1). et al., 2007). These are involved in DMSP transport To identify the relevant genes in these new (dddT), regulation (dddR) or in the later stages of isolates, genomic libraries of Psychrobacter J466 DMSP catabolism through dddB and dddC, which respectively encode polypeptides related to alcohol 9000 dehydrogenases and aldehyde dehydrogenases (Figure 2). Psychrobacter J466 also has a gene that 8000 encodes a dehydratase-family member (Pfam00378). 7000 Pseudomonas J465 has a gene encoding an IclR-type regulator that is likely co-transcribed with dddT, 6000 dddB, dddC and dddR, and a gene, possibly co- 5000 transcribed with dddD, which may encode a major

g protein / hour 4000 facilitator transporter. µ In a direct study of DMSP turnover in Atlantic 3000 Menhaden (Brevoortia tyrannus), a close relative of 2000 the Herring, approximately 65% of the DMSP was 1000 incorporated into the animals’ flesh or was excreted, nmol DMS / unmodified, with very little DMS being released when 0 Pseudomonas J465 Psychrobacter J466 fish were fed DMSP-containing algae (Hill and Dacey, 2006). The fate of the remaining 35% is unknown, Figure 1 Dimethyl sulfide (DMS) production in Pseudomonas but could be due to bacterial catabolism in one of J465 and Psychrobacter J466. Cells were pre-grown with 5-mM two ways. First, the DMS formed by Ddd þ bacteria dimethylsulfoniopropionate (DMSP) (grey), acrylate (striped), 3-hydroxy-propionate (3-OH-P) (stippled), or no inducer (white). may be catabolized by other bacteria that use DMS DMS production is in nmol DMS per mg protein per hour ( Â 105) as a substrate (Scha¨fer, 2007). Second, another route (mean of two independent assays). for DMSP catabolism involves DMSP demethylation,

49% 36% 51% 77% 63% 1kb Psychrobacter J466 CoA dddT dddD dddC dddB hydratase

46% 70% 73% 75% 86% 70% 62% Pseudomonas J465 MFS transporter dddD dddT dddB dddC iclR dddR

Marinomonas MWYL1 dddD dddT dddB dddC dddR

Figure 2 Arrangement of ddd genes in Pseudomonas J465, Psychrobacter J466 and Marinomonas MWYL1. Genes previously identified as being involved in dimethylsulfoniopropionate (DMSP) catabolism (ddd) or that may be in the same transcriptional unit as ddd genes are shown. The percentage identities of gene products to those of the corresponding genes in Marinomonas MWYL1 for dddT, dddD, dddC, dddB and dddR are shown, as are those of a CoA hydratase to that of Brevibacillus brevis NBRC 100599, of iclR to that of Colwellia psychrerythraea 34H and of the major facilitator transporter to that of marine g-proteobacterium HTCC2207. The dashed vertical line indicates a breakpoint in dddB in the Psychrobacter library cosmid. Sequences are deposited at Genbank with accession numbers GQ370383, GQ370384, GQ370385 and GQ370386.

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